Characterization of Metabolites in DomesticSow Urine After Intravenous Administration of Radioactive Estrogenand CorticosteroidsI PATRICIA JONES-WITTERS, MARY J. HURLEY*, REBECCA PHILLIPS ~ Department of Zoology, Ohio University, Athens 45701 B. L. BROWN 4 and R. E. ERB Department of Animal Sciences, Purdue University West Lafayette, IN 47907
chromatographic properties of tetrahydroeorticosterone and corticosterone. Considerable radioactivity from injection of both corticoids was isolated in the cortol, cortolone, and ll-ketoetiocholanol o n e - llfl-hydroxyetiocholanolone areas of chromatograms. The data for corticolds agree with similar data for the human being and cow.
Abstract
We measured distribution of radioactivity among urinary metabolites excreted in nonpregnant and ovariectomized sows after intravenous injection of radionuelides (1'4carbon) labeled estrone, estradiol-17fl, cortisol, and corticosterone. Treatment with an enzyme preparaffon (Glusulase) containing both/3-glucuronidase and sulfatase activity, rendered .~tractable over 95% of the radioactivity recovered from urine with diethyl ether (estrogens) and ethyl acetate (co~coids). Only an additional 1 to 4% cff the radioactivity was extracted following solvolysis of the aqueous residue remaining after enzyme hydrolysis and extraction. Radioactivity in nonpregnant sow urine was predominantly in the estrone fraction following injection of either estrone or estradiol-17fl. Moreover, the principal metabolite was estrone monoglucuronide. Only traces of estradiol-17fl and an estriol-like compound were detected. Two other isolates contained radioactivity. One compound probably was 2mcthoxyestrone, but structure of the other compound (X1) could not be ,established. The principal urinary metabolites from injection of cortisol eo~responded to chromatographic properties of tetrahydrocortisol and tetrahydrocortisone. Both metabolites were low in urine following injection of corticosterone. The major urinary metabolites from corticosterone iniecfion corresponded to
Introduction
In contrast to the cow (18, 33, 35) the domestic sow excretes in urine a major portion of 14C-metabolites of intravenously injected x'Clabeled progesterone (24), estrone, and estradiol-17fl (4, 29) and eortisol and corticosterone (4). Although sow urine contains nearly all of the radioactivity excreted after intravenous injection of 14C-labeled estrone, estradiol-17fi (4, 29), cortisol, and corticosterone (4), only urinary estrone has been established clearly as resulting from estrogen metabolism (31). The purpose of our study was to advance current knowledge for the sow concerning characterization of major urinary metabolites of in vivo metabolized ~4C-labeled estrone, estradiol-17fl, cortisol, and corticosterone. A brief report has characterized l~C-labeled urinary estrogens (15). Materials and Methods
General. Two yearling Yorkshire-Hampshire crossbred sows, one intact (137 kg) and one ovariectomized (160 kg), were used. The sows were housed in a heated and mechanically ventilated building ~o,r 6 wk prior to the experiment. The intact sow was injected intravenously with four different z4C-steroids (New Received May 31, 1974. 1 Journal Paper 5512, Purdue University Agri- England Nuclear Corp.) each labeled at the cultural Experiment Station, West Lafayette, IN --4-C position. These were estradiol-17fl (2.26 47907. /zCi; 13.66 /zg), estrone (2.12 /zCi; 12.34 /zg) Present address: University of Michigan School 6 wk later: corticosterone (4.74/zCi; 29.37/zg) of Medicine, Ann Arbor 48103. 8Present address: The Ohio State University, 5 me later: and cort£sol (4.41 /zCi; 30.79 /~g) 3 wk later. The ovariectomized sow was dosed Columbus 43210 4Present address: ]3iologieal Science Research with z4C-cortisol (4.25 /zCi; 29.69 /~g) 4 me after ovarieetomy. Radioactivity in urine was Unit, FBI Laboratory, Washington, DC. 20530. 41
42
J O N E S - W I T T E R S ET AL
indistinguishable from background by 7 days after treatment. Techniques for proof of purity of the hormones, dosing, and rates of excretion in urine have been reported for these injections (4). Urine, collected by urinary catheter, was processed as described by Edgerton ,and Erb (7) and stored at --20 C until analysis. Only urine collected during the fi~rst 0 to 24 h after dosing was used for isolating labeled compounds. General approaches to extraction of free and conjugated steroid metabolites from sow urine, subsequent partial purification and isolation of compounds, and methods of quantification have been published (13, 14, 25, 32). Hydrolyzed urinary estrogen. Urine collected for the Rrst 4 h following injection of estrone and the first 12 h ~ollowing injection of estradiol-17fl was subjected to three successive treatments; enzyme, solvolysis, and acid, to cleave the conjugates from the steroidal aglycons (14). Diethyl ether extraction procedures (4) and separation of extracted compounds into enzyme-neutral, enzyme-phenolic (30), solvolysis-neutral, solvolysis-phenolie, acid-neutral, and acid-phenolic (14) ~ractions was as previously described. Estrogens freed by hydrolysis were separated by thin layer chromatography (TLC) System I which was methanol-methylene chloride (1:20, vol/vol). Location of standards, elution of unknowns &ore zones corresponding to standards, and concentration of metabolites were as previously described (25). Prior to derivative formation the extracts stored in ethanol (--10 C), were dried under vacuum, and the residue was transferred with freshly distilled tetra~ydrofltran (3 X 200/~1) into 1 ml conical tubes. Then the solvent was evaporated under a stream of nitrogen. O-methyloxime (1) and trimethylsilyl ether (13) derivatives were prepared as well as double derivative. For the latter, O-methyloxime was prepared first, the reagent removed with a stream of CO2 with the reaction tube placed in a warm water bath, and then the trimethylsilyl ether (TMS) was prepared. Radioactive compounds were eluted from a gas-liquid chromatography (GLC) column with a 50-50 stream splitter. Steel columns (368 X .32 ern) were packed with 2% liquid phases OV-1 and OV-225 (Supelco, Inc.) coated on Anakrom ABS (80-90 mesh, Analabs, Inc.). Nitrogen and hydrogen flow rates were 25 ml/min. Analytical GI_,C was with a dual column instrument (Varian Aerograph, Model 204-1C). Rentention times were deterJOURNAL OF DAIRY SCIENCE VOL. 58, N o . l
mined relative to 5~-cholestane. A combination gas chromatograph-mass spectrometer (PerkinElmer, Model 270) was used to obtain mass spectral data. Urinary estrogen conjugates. Urine collected from 0 to 8 h after rdC-estrone injection was pooled a~d six 25-ml aliquots were designated samples A through F. Counting aliquots were taken at each step of the procedure to isolate the conjugated estrogens. Free estrogens were extracted from unhydrolyzed or hydrolyzed urine with diethyl ether (1:1, vol/vol). Conjugated estrogens were removed from urine with Amberlite XAD-2 resin (Robin and Haas Co.) by the procedures of Bradlow (2) and Osawa and Slaunwhite (21). Estrogen glucuronides were freed from their conjugating moiety with a fl-glueuronidase (Ketodase, Warner-Chflcott) which does not have sulfatase activity. Glass plates (20 X 20 can) coated with silica gel G (25 mm thick) and three TLC systems were used to separate tmconjugated (System I identified in the preceding section) and conjugated estrogens (Systems II and III). The mixture of conjugates from the XAD-2 resin column were separated in System II (ehloroform:isopropyl ,alcohol:formic acid; 100:60:30, vol/vol/vol) into sulfate, anonoglucuronide, and diconjugate zones. The monoglucuronides were separated in System III into estriol glucuronide and estradiol-estrone glueuronide zones by developing twice in chloroform: methanol: acetic acid (8:1:1, vol/ vol/vol) (27). Mter development, reference s~andards were located (26) and corresponding zones of unknowns were marked on the ehromatograms. Then the silica gel in these zones was scraped onto glassine paper, transferred to test tubes, and 10 ml of solvent was added. The mixture was allowed to stand overnight before filtering through a sintered glass funnel. Chloroform-methanol (1:1 vol/vol) and 50% aqueous methanol were used to elute u nconjugated and conjugated estrogens, respectively. Enzymatic hydrolysis of conjugates and GLC procedures were as reported in the preceding section. Urinary corticold metabolites. Urine voided within 24 h after injection of either x*C-cortisol or 14C-cortieosterone was used to extract the urinary ~*C-labeled metabolites. The procedures initially followed were like those reported for cow urine by Willett et al. (32) except for two differences. During extraction the solvolysis step was unnecessary when fl-glueuronidase (Glusulase, End9 Laboratories) was used for initial hydrolysis. Also, colu~mn chromatography was un-
S T E R O I D METABOLITES
43
TABLE 1. Distribution of extracted radioactivity (~) within sequential cleavage treatments and fractions from urine following injections of a4C-labe]ed estrone and estradiol-17/~. I-lxnrnone injected
Sequential treatment
Fraction Acid"
Phenolic
Neutral
(~) Estrone
Enzymeb Solvolysis
65.3 _ 4.6 ~ .23 _ .05
36.1 - 2.0 background
2.5 ± .3 .24d
Estradiol-17/~
Enzyme Solvolysis
53.3 3.2
38.5 ± 2.4 background
1.4 - .2 .16 ± .02
___ 4.1 ± .66
Extracted into the NaOH/NaCL wash which was at pH 13. Adiustment to pH 3 in subsequent samples resulted in extraction of 79?o of the radioactivity in the acid fraction as result of partitioning at pH 13. b Glusulase, Endo Laboratories. Standard error. d Samples were pooled. necessary as an initial step to separate 14C-labeled compounds due to smaller quantities of pigments in sow urine extracts compared to cow urine extracts. Paper chromatography systems A, C, and D as identified and diagrammed by Willett et ,al. (32) were used to separate urinary radioactive metabolites of aaC-labeled cortisol and cortic~>sterone. Chromatograms of unknowns were observed under UV light to detect a,fl uasaturated ketone structures. Blue tetrazolium and phosphomolybdic acid were used to test for side chains and reactive groups (5). Results and Discussion
Metabolites of estrogen. Totals of 81% and 91% of the radioactivity in urine from injection of ~ C estrone and estradiol-17fi, respectively, were extracted by sequential hydrolysis. Enzyme treatment .alone rendered extractable with diethyl ether, 99.7 ___ 4.3% and 95.4 ± 8.0% of the urinary radioactivity (x of six extractioa~'s) recovered for the estrone and estradiol-17fl iniectious. Due to efficiency of hydrolysis of the crude enzyme mixture, Ghisulase, which contained both sulfatase and ghicuronidase activity, only .31 ± .01% and 4.6 ± .4% of the radioactivity was extracted following solvolysis of urine from the estrone and estradiol-17fl injections. These percentages are s'mailar to those repol~ed by Tarqui et al. (29). The increased amount of r~dioactivity extracted after solvolysis of urine from the estradiol17fl iniection as compared to estrone may represent a minor difference in conjugation pathways between estrone and estradiol-17fi. No additional detectable radioactivity was ether extractable after acid hydrolysh. Thus, it may be unnecessary to use other than Glusulase on
nonpregn.ant sow urine to render estrogens ether extractable. This, however, would need to be checked on additional individuals at different periods of the estrous cycle and during pregnancy to determine the extent of variation between a~_imals. Diethyl ether extracts of urine following enzyme hydrolysis contained more radioactivity in the phenolic fraction than in the acid fraction (Table 1). In eorapaa'ison, enly trace amounts w e r e in the neutral fraction. The data for urine following estradioI-I7fl injection were similar to data for estrone except 3.2% instead of .23% of the radioactivity was in the solvolysis-phenolle fraction (Table 1) which suggests the presence of a sulfate conjugate resistant to Ghisulase. The presence of 36 to 38% of the TABLE 2. Distribution of radioactivity after thinLayer ebxomatography (TLC) of the enzyme-acidic fraction from urine following injection of a4C_,labeled estrone. Collection period
TLC zone system I ~
(h)
(Standard)
0 to 8 pool
Estrone Estradiol-17~ Estriol
4 to 5 pool Estrone Estradiol-17~ Estriol
Radioactivity (No)
(~;)
I
2. Q
II III IV V
36.6 1.7 54.3 4.6
I II III IV V
7.4 15.1 12.4 50.9 14.1
"Zones were numbered starting at the solvent front; 20 X 20 cm plate chromatographed in methanol:methylene chloride ( 1: 9.0, vol/vol). JOURNAL OF D/dRY SCIENCE VOL. 58, NO. I
44
JONES-WlTTERS ET AL
total ~C extracted in the base wash aqueous layer (acidic fraction) was unanticipated and indicated compounds more polar than estriol (19). However, a major portion (79%) of the radioactivity in the acid ~raction was extracted with diethyl ether in subsequent samples after adjusting the NaOH/NaC1 wash from pH 13 to pH 3. About 50% of the radioactivity remaining in the enzyme-acidic fraction a~ter extraction with diethyl ether at pH 3 was in the estriol fraction (Table 2). Approximately 97% of the radioactivity in the enzyme-phenolic fraction (fl~rst six samples extracted) was in the estrone TLC zone (System I) following either estrone or estradiol-17/3 injection. The relatively small .amount of radioactivity in the solvolysis-phenolie fraction from the estrone injection was 100% in the estrone TLC zone. The comparable estradiol-17/3 data showed that, in addition to 91% the radioactivity in the estrone TLC zone, 4.4% and 4.7% of the radioactivity was at the origin and in the estradiol-17fl TLC zone. Samples of the estrone TLC zone from both estrone and estradiol-17fi injections were chromatographed on GLC as trimethylsilyl ether derivatives, and the column effluent was collected for measurement of radioactivity. The estrone peak contained 67% of the radioactivity. The remaining radioactivity, especially in the extract following estrone injection, may have been s',nnilar to an unknown compound previously designated X1 by Jones (12). Even though attempts to identify this compound were unsuccessful, the compound was not a contaminant of r'C-labeled compounds injected. Another unidentified peak, accounting for 10% of the radioactivity chromatographed, was eluted from urinary extracts following injection of estrone but not estradiol. As will be shown later, 21% of the radioactivity was in the same peak following fl-glucuronidase treatment and TLC (System III) of the phenolic glucuronide fraction of extract from the estrone injection. The compound may be the 2-methoxyestrone identified by Terqui (28). Mass spectra were obtained of standard estrone TMS, and of the TMS derivatives of endogenous estrone and X1 isolated from extracts of urine after estrone injection as each of the compounds ~vas eluted from an OV-1/OV-225 column. The spectrum of endogenous estrone correlated with standard estrone and with published spectra (20). The spectrum of X1 TMS could not be identi~ed as a steroid. In associated work on pregnant sows, Edgerton and Erb (7) reported one unknown comJOURNAL OF DAIRY SCIENCE VOL. ~8, NO. 1
pound identical in GLC properties to estradiol17fl and another unknown with GLC properties similar to estradiol-17~. Mass spectral analysis of the estradiol TLC zone of day 103 pregnancy urine from the sows mentioned above verified the GLC identifi~Cation of estradiol-17fl by Edgerton and Erb (~/). Comparison of the acetylated total phenolic extract (7) with acetylated TLC zone extracts indicated that X1 probably was the compound with GLC properties similar to the acetate derivative of standard estradiol-17a. Urinary estrogen con/ugates. Each of the six 25-ml replicate samples contained 19,100 __ 379 dpm. The distribution of radioactivity after extraction of the urine with diethyl ether showed that 95.0 __ .9% of the labeled estrone either had been conjugated or metabolized and conjugated by the sow and remained in the aqueous layer. Only 1.8 ± .2% was ether extractable prior to hydrolysis as compared to 14.5% from urine of ,a pregnant sow (16). The lower value may reflect differences in physiological state and dosage. Estrogen conjugates are compounds of the type absorbed by XAD-2 resin which can be eluted by the polar alcohols, ethanol and methanol. Almost all of the r~dioactivity (91.8 ± 1.1%) was in these conjugates. The first fraction, eluted with ethanol, contained 71.1 ± 2.2% of absorbed radioactivity; and the second fraction, eluted with methanol, contained 22.9 ± 1.2%. The remainder was essentially accounted ~or in the water wash. When these two alcoholic fractions were processed separately, the percentage distribution of radioactivity by TLC zone following chromatography of conjugated and nonconjugated compounds was almost identical. Therefore, the ethanol and methanol fractions were combined .as the alcoholic fraction for further analyses. After the alcoholic extracts from Sample C were hydrolyzed with Ketodase to free the estrogens present as glucuronides, 96 ± 3% of the 1~C was extracted with diethyl ether. The compounds extracted into ether were chromatographed in System I. The estrone zone contained 84.4% of the radioactivity, and ,approximately 9% of the total dpm were in the estriol and adjacent less polar TLC zones. Alcoholic fraction,s from samples A, B, and D to F contained 93.3 +__ 2.1% of the ~*C in the monoglucuronide zone and only 5.6 ± 2.1% in the diconjugate zone (Table 3). Only .8 ± .2% of the 14C was in the sulfate zone and only .3 ± .1% at the origin. These results are
S T E R O I D METABOLITES
45
TABLE 3. Distribution of radioactivity (%) folio,wing thin-layer chromatography (System II) ~ of the conjugates eluted from amberlite XAD-2 by ethanol and methanol.
Sample
Zone I (origin)
Zone II ( diconiugates )
A B D E F Mean
.2 .5 .2 .6 .2 .3 - .1b
1.2 1.5 4.2 10.2 10.8 5.6 ± 2.1
Zone III (monoglucuronides) 98.2 97.2 94.1 88.2 88.6 93.3 + 2.1
Zone IV (monosulfates, solvent front) .4 .8 1.5 1.0 .4 .8 _-2 .2
Chloroform:isopropyl alcohol:formic acid ( 100:60:30 ), ( vol/vol/vol ). b Standard error. in marked contrast to those of Kubomichi (16) who reported 21.2% of the total estrogens in the sulfate fraction 'and 64.3% in the glucuronide fraction. However, the sulfate fraction was obtained by acid hydrolysis at 32 C with simultaneous extraction into ether, and the glueuronide fraction by vigorous ,acid hydrolysis after removal of the sulfates. These methods have caused structural alterations, and Kubomichi (16) recommended that future research use enzymatic rather than acidic hydrolysis. The monogtueuronide zone from Sample B was rechromatographed in System Ili. The estriol-3-glucuronide zone contained 8.2%, and the estradiol-17-glueuronide-estrone-3-glucuronide zone contained 87.6% of the radioactivity. When the TLC monoglucuronide zones from Samples D and E were hydrolyzed by Ketodase and extracted with diethy1 ether, more than 98% of the radioactivity was in the organic phase. When the concentrated diethyl ether extracts were chromatographed in System I, 94.2 4- 1.0% of the label was in the estrone zone, 2.1 ___ 0.6% was in the estriol zone, and 1.9 ± 0.1% was in the estr~diol zone. The TMS derivatives of compounds: in the estrone TLC zone from Samples D and E above were chromatographed by GLC as described earlier. Four peaks were resolved from the biological material. The relative retention
times (RRT) of these peaks were based on 5~eholestane, which had a retention time of 15.2 rain. Of the radioactivity ehromatographed, peak 1, .an unknown (RRT .52), contained 8.1%; peak 3, estrone (RRT .80), contained 65.4%; and peak 4, an unknown (RRT .89), contained 21.3% of the radioactivity chromatographed. These results are in fair agreement with results cited in the preceding section for estrogens extracted after hydrolysis and separated in the same TLC system. Peak 4 may be 2-methoxyestrone, ~dentit%d in the urine of the pregnant sow following injection of labeled estrone and estradiol (28). Urinary corticoid metabolites. The percentage ef 14C extracted with ethyl ,acetate before enzyme hydrolysis accounted for only 4.6 to 9.4% of the total radioactivity (Table 4). More than 60% was extracted following enzyme hydrolysis (Glusulase) of urine from cortisol injections as compared to about 50% for cortieosterone. From 16 to 28% of the radioactivity was not extracted, and an additional 9 to 17% was unaccountable loss. As shown from preliminary extractions, only an additional 1 to 4% could have been extracted by including solvolysis as used by Willett et al. (32). Radioactivity extracted before enzyme hydrolysis and after solvolysis was too low ~or characterization of compounds in the respective extracts.
TABLE 4. Average percentage of 14C extracted before and after enzyme hydrolysis of sow urine voided after injection of ~4C eortisol and cortieoateroneL Sow Ovariectomized Intact Intact
14 C compound Cortisol Cortisol Cortieosterone
Before
Hydrolysis After
Total extracted
4.6 ± .4b 6.9 ± .6 9.4 ± 2.0
69.9 ± 1.9 60.9 ± 2.6 49.4 --- 3.4
74.5 --- 2.8 67.8 ± 3.1 58.8 ± 3.1
* Each sample prior to extraction contained approximately 1.0 X 105 dpm. ~' Standard error; n = 12. JOURNAL OF DAIRy SCIENCE VOL. 58, NO. 1
46
JONES-WITTERS ET AL
TABLE 5. Distribution of extracted a~C (average ~) among urinary corticoid rnetabolites isolated by paper chromatography systems A, C, and D as described by Willett et al. (32). Compounds ~
Ovariectomized Cortisol
Intact Cortisol
Cortieosterono
(~) 6 hydroxycortisol Cortol Cortolone Tetrahydroeortisol Tetrahydrocortisone Cortisol Corticosterone Tetrahydroeortieosterone 11-ketoetiocholanolone and 11/S-hydroxyetiocholanolone
2.2 11.6 21.4 23.8 30.8 5.9 .3 3.4
_.+ _ _-. __. ± ± --±
13.0 ±
1.3~ 4.9 3.0 4.1 10.7 2.9 .2 1.0 2.4
7.0 13.5 11.7 43.2 8.8 6.9 .7 .5
__+ 2.9 ___ 2.8 _ 2.0 ± 9.2 __. 2.0 +__ 3.3 ± .7 _ .3
8.6 ± 3.9
1.3 11.7 5.6 2.7 7.7 0.5
--+ .9 ± 4.6 _ 1.9 + 1.~ ± 7.1 - .3
20.5 +-. 3.7
31.6 ± 8.2 18.5 ± 5.0
Standard error; n = 9. A minimum of nine compounds was isolated from extracts of enzyme hydrolyzed urine (Table 5). The maior urinary metabolites of cortisol corresponded to chromatographic properties of tetrahydrocortisol (THF) and tetrahydroeortisone (THE) standards. Although THF was the major metabolite in the intact sow, totals of T H F and THE were similar, averaging 54.6% and 52.0% for the ovariectomized and intact sows and similar to data for human beLugs (11, 23). In contrast, both T H F and T H E were low in urine of the intact sow following injection of corticosterone. For the corticosterone injection, major urinary rnetabolites corresponded to the isolated eorticosterene and tetrahydrocorticosterone (THB) standards. These two compounds ,accounted ~or 52% of the radioactivity isolated from extracts of urine from the eortieosterone injection. These results agree with similar data for human beings (22) and cows (32). The urine of both sows contained considerable radioactivity in areas of chromatogrr~ms corresponding to cortol, cortolone, and ll-ketoetiocholanolone-llfl-hydroxyetiocholanolone standards (Table 5). General Discussion
Estrogens. The estrone ~raction of urine from the sow contains the largest amount of estrogen metabolite, and the amount of estradiol17/3 is low .as previously reported (17, 29, 31). Additionally, the loss of a large percentage of radioactivity not in estrene or estradiol-17fl fractions ,agrees with Terqui et a]. (29). The evidence, although inconclusive, favors a small a~ount of an estriol-like compound in sow urine which also tras been reported by others
(3, 19, 28). In the nonpregnant sow estrone is eonjugatJouaNaL OF DAIRYSCI~NCSVOL. 38. NO. 1
ed primarily as the monoglucuronide (Table 3). Terqui et al. (29), using the Brown method of separation, found that when a sow was injected with either labeled estrone or estradiol, the bulk of the label was recovered in the urine in the estrone fraction. Lunaas (17), also using Brown's method of separation, found that estrone was the major estrogen recoverable ~rom sow urine, both during early pregnancy and during the estrons cycle. However, he discarded the portion of urine extract which would have contained estriol. Kubomichi (16) and Bredeck and Mayer (3) reported that estriol was the major urinary estrogen in pregrrant sows in contrast to the 98% estrone reported by Edgerton and Erb (7) for day 110 of pregnancy. Edgerton and Erb (7) used enzyme hydrolysis during extraction and quantification by gas liquid chromatography, as compared to acid hydrolysis and quantification by colorimetry (3, 16). Kubomichi (16) attempted to identify the types of conjugates by differential acid hydrolysis. He found that 83% of the estrogens were conjugated as ghicuronides and 21% were conjugated as sulfates. This does not agree with our ~ndings since over 95% of the 1'4C was in the glucuronide fraction. However, differences in conjugate isolation technique and reproductive status of the sows probably account for the different results. Incomplete hydrolysis and desta'uction of aglycones may be avoided by extraction of the estrogen conjugates with XAD-2 resin rather than by differential acid hydrolysis (21). Dtffour and Raeside (6) have suggested that estrogen formation occurs largely in the placenta of the pregnant sow, so that the percentage estrogen conjugated ,as sulfate may well differ from that in urine of the nonpreg-
STEROID METABOLITES
nant so~v. If it is later verified in a larger population of sows that urinary estrone of sows is conjugated mostly as the monoghmuronide, then quantitative methods can be greatly simplified by using only enzyme hydrolysis and substitution of radioimmunoassay for GLC quanti~cation. Corticoids. T h e sow excretes in urine 80 to 90% of the radioactivity injected as zdC-labeled cortisol and corticosterone (4) as compared to 20 to 29% for the cow (33). The majority of corticoid metabolites in sow urine were similar to the parent compounds relative to containing 21 carbons (9) and were reduced in the A ring to tetrahydro derivatives (10) of THF, THE, and THB. The presence of a larger quantity of T H E (30.8%) than T H F (23.8%) in urine of the ovariectomized sow (Table, 5) was about the same as reported for the human being (9, 9,3). The predominance of T H F (43.2%) rather than T H E (8.8%) in the urine of the intact sow after eortisol injection was similar to changes associated with disease induced stress in the human being (11). Therefore, the intact sow may have had a stress reaction when cortisol was administered. Willett and Erb (34) observed a marked inerease in plasma corticoids when 14C-labeled cortisol was adininistered to a heifer. Also, plasma cortisol was increased in heifers as much as five times from psychological stimuli (34). In general, it appears that the primary urinary route of excretion (4) and the major urinary metabolites of cortisol and corticosterone (Table 5) in the sow are typical of results for the human being (9, 11, 22, 23). Acknowledgments The authors thank L. A. Edgerton, Smith Kline Laboratories, £or administration of estrogens and collection of urine while at Purdue University; Philip Gill, E. I. du Pont de Nemours and Co., for gas chromatography-mass spectrometry, W. W. l~audler, Department of Chemistry, Ohio University, for interpretation of the mass spectra; and W. C. Afford and W. Klyne for sodium estradiol-3-glueuronide, sodium estradiol-17fl-glucuronide, sodium estriol3-glucuronide, sodium estriol-18a-glucuronide, sodium estriol-17fl-glucuronide, 16a-hydroxyestrone- 16~-glucuronide, 16fl-hydroxyestrone16fl-glucuronide, and sodium estrone-3-glucuronide from the MRC Steroid Reference Collection. References (1) Allen, J. G., C. H. Thomas, C. J. W. Brooks, ,and B. A. Knights. 1969. The determina-
47
tion of stereochemistry at C-5 of 3, 6-dioxygermted steroids. Steroids 13:133. (2) Bradlow, It. L. 1968. Extraction of steroid conjugates with neutral resins. Steroids 11: 265. (3) Bredeck, H. E., and D. T. Mayer. 1958. Estrogenic steroids in swine pregnancy urine. Page 157 in E. X. Gassner, ed., Reproduction and infertility, III Symposinrn. Pergamon Press, New York, NY. (4) Brown, B. L., L. A. Edgerton, L. B. Willett, R. D. Randel, T. G. Dunn, and R. E. Erb. 1970. Excretion of 14C in urine of the domestic sow after inieetion of radioactive estradiol-17fl, estrone, cortieosterone and cortisol. J. Anita. Sci. 31:1186. (5) Domingnez, O. V. 1967. Chromatography of steroids on paper. Page 135 in H. Carstensen, ed., Steroid hormone analysis, Vol. 1. Marcel I)ekker, Inc., New York, NY. (6) Dufour, J., and J. I. Raeside. 1969. Hydroxysteroid dehydrogenase activity in the placenta of the domestic pig. Endocrinology 84:426. (7) Edgerton, L. A., and R. E. Erb. 1971. Metabelites of progesterone and estrogen in domestie sow urine. I. Effect of pregnancy. J. Anita. Sci. 32:515. (8) Frantz, A. G., F. H. Katz, and J. W. Tater. 1961. 6/3-hydroxycortisol and ocher polar corticosteroids: Measurement and significance in human urine. J. Clin. Endoerinol. Metab. 21:1290. (9) Fukushima, D. K., H. L. Bradlow, L. Hellman, B. Zumoff, and T. F. Gallagher. 1960. Metabolic transformation of hydrocortisone4JdC in nonnal men. J. Biol. Chem. 235: 2246. (10) Gold, N. I. 1961. Partial chara~evizatlon of the metabolites of cortisol-4-1~C in the dog. I. The intact dog. J. Biol. Chem. 236:1924. (11) Gray, C. H., and D. A. Shaw. 1965. The metabolism of (4-~'C) cortisol in patients with collagen diseases. J. Endocrinol. 33:33. (12) Jones~ P. H. 1967. Isolation, identification ,and measurement of metabolities of progestins and estrogens in the urine of donaestie sows during the estrous cycle. Ph.D. Thesis. Purdue University, West Lafayette, IN. (13) Jones, P. H., and R. E. Erb. 1967. Modified procedure for estimating estrogens in urine. J. Dairy Sci. 58:772. (14) Jones, P. H., and B. E. Erb, 1968. Comparison of metabolites of progesterone-14J~C in urine from a nonprognant domestic sow. J. Anita. Sci. 27:1049. (15) Jones-Witters, P., and R. Phillips. 1971. Metabolites of ~C-labeled estrogens in the sow. J. Anita. Sci. 33:1159. (Abstr.) (16) Kubornichi, M. 1966. Studies on the m-inary estrogens of pregnant sows. Nat. Inst. Anita. Health Quart. 6:33. JOURNAL OF DAIRY SCIENCI~ VOL. ~8, NO. 1
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JONES-WITTERS E T AL
(17) Lunaas, T. 1962. Urinary estrogen levels in the sow during estrous cycle and early pregnancy. J. Reprod. Fert. 4:13. (18) Mellin, T. N., and R. E. Erb. 1966. Estrogen metabolism and excretion during the bovine estrous cycle. Steroids 7:589. (19) Mellin, T. N., R. E. Erb, and V. L. Ester,green. 1965. Quantitative estimation and identification of estrogens in bovine urine. J. Dairy Sci. 48:895. (20) Okerholm, R. A., S. I. Clark, and H. H. Wotiz. 1971. Effect of derivative formation on the mass spectra of the estrogens. Anal. Biochem. 44:1. (21) Osawa, Y., and W. R. Slaunwhite, Jr. 1970. Studies on phenolic steroids in human subjects. XIII. A rapid assay of urinary conjugates in pregnancy. Steroids 15:73. (22) Peterson, B. E., and C. E. Pierce. 1960. The metabolism of eortieosterone in man. J. Clin. Invest. 39:741. (23) Romanoff, L. P., C. W. Morris, P. Welch, R. M. Rodriquez, and G. Pineus. 1961. The metabolism of cortisol-4-1'C in young and elderly men. L Secretion rate of eortiso.1 and daily excretion of tetrahydroeortisol, allotetrahydroeortisol, tetrahydroeortisone and cortolone (20a and 20~). J. Clin. Endoerinol. Metab. 21:1413. (24) Schomberg, D. W., W. R. Featherston, and R. E. Erb. 1965. Excretion of metabolites of progesterone-4-a~C in the cycling sow. J. Anim. Sei. 27:1195. (25) Sehomberg, D. W., P. H. Jones, W. R. Featherston, and R. E. Erb. 1966. Identification of metabolites of progesterone-4-~4C in domestic sow urine. Steroids 8:277.
JOURNAL OF DAIRY SCIENCE VOL. 58, N O . I
(26) Stahl, E., ed. 1965. Thin-layer chromatography. Academic Press, Inc., New York, NY. (27) Takriti, N., and W. Thorn. 1965. Separation and detection of gluenronides. Angew. Chem. Intern. Ed. Eng. 4:1095. (28) Terqui, M. 1971. Estrogen metabo//sm in the pregnant sow. I. Urinary estrogens. Ann. Biol. Anita. Biochem. Biophys. 11:569. (29) Terqui, M., P. Rombauts, and J. Fevre. 1968. Comparative fecal and urinary excretion of estrogens in the ewe and sow. Ann. Biol. Anita. Biochim. Biophys. 8:339. (30) TiUson, S. A., R. E. Erb, and G. D. Niswender. 1970. Comparison of luteinizing hormone and progesterone in blood and metabolites of progesterone in urine of domestie sows during the estrous cycle and early pregnancy. J. Anita. Sei. 30:795. (31) Velle, W. 1959. Isolation of oestrone from the tu-ine of the pregnant sow. Aeta. Vet. Scan& 1:19. (32) WiUett, L. B., B. L. Brown, and R. E. Erb. 1972. Isolation of metabolites of 4-a4C-cortieosteroids from urine of an ovarieetomized heifer. J. Dairy Sci. 55:65. (33) Willett, L. B., T. G. Dunn, B. L. Brown, and R. E. Erb. 1971. Excretion of 4-~C eortieosteroids in urine by an ovarieetomized bovine. J- Anita. Sci. 32:119. (34) Willett, L. B., and R. E. Erb. 1972. Short term changes in plasma cortieoids in dairy cattle. J. Anita. Sci. 34:103. (35) Williams, W. F. 1962. Excretion of progesterone and its metabolites in milk, urine and feces. J. Dairy Sei. 45:1541.
chromatographic properties of tetrahydroeorticosterone and corticosterone. Considerable radioactivity from injection of both corticoids was isolated in the cortol, cortolone, and ll-ketoetiocholanol o n e - llfl-hydroxyetiocholanolone areas of chromatograms. The data for corticolds agree with similar data for the human being and cow.
Abstract
We measured distribution of radioactivity among urinary metabolites excreted in nonpregnant and ovariectomized sows after intravenous injection of radionuelides (1'4carbon) labeled estrone, estradiol-17fl, cortisol, and corticosterone. Treatment with an enzyme preparaffon (Glusulase) containing both/3-glucuronidase and sulfatase activity, rendered .~tractable over 95% of the radioactivity recovered from urine with diethyl ether (estrogens) and ethyl acetate (co~coids). Only an additional 1 to 4% cff the radioactivity was extracted following solvolysis of the aqueous residue remaining after enzyme hydrolysis and extraction. Radioactivity in nonpregnant sow urine was predominantly in the estrone fraction following injection of either estrone or estradiol-17fl. Moreover, the principal metabolite was estrone monoglucuronide. Only traces of estradiol-17fl and an estriol-like compound were detected. Two other isolates contained radioactivity. One compound probably was 2mcthoxyestrone, but structure of the other compound (X1) could not be ,established. The principal urinary metabolites from injection of cortisol eo~responded to chromatographic properties of tetrahydrocortisol and tetrahydrocortisone. Both metabolites were low in urine following injection of corticosterone. The major urinary metabolites from corticosterone iniecfion corresponded to
Introduction
In contrast to the cow (18, 33, 35) the domestic sow excretes in urine a major portion of 14C-metabolites of intravenously injected x'Clabeled progesterone (24), estrone, and estradiol-17fl (4, 29) and eortisol and corticosterone (4). Although sow urine contains nearly all of the radioactivity excreted after intravenous injection of 14C-labeled estrone, estradiol-17fi (4, 29), cortisol, and corticosterone (4), only urinary estrone has been established clearly as resulting from estrogen metabolism (31). The purpose of our study was to advance current knowledge for the sow concerning characterization of major urinary metabolites of in vivo metabolized ~4C-labeled estrone, estradiol-17fl, cortisol, and corticosterone. A brief report has characterized l~C-labeled urinary estrogens (15). Materials and Methods
General. Two yearling Yorkshire-Hampshire crossbred sows, one intact (137 kg) and one ovariectomized (160 kg), were used. The sows were housed in a heated and mechanically ventilated building ~o,r 6 wk prior to the experiment. The intact sow was injected intravenously with four different z4C-steroids (New Received May 31, 1974. 1 Journal Paper 5512, Purdue University Agri- England Nuclear Corp.) each labeled at the cultural Experiment Station, West Lafayette, IN --4-C position. These were estradiol-17fl (2.26 47907. /zCi; 13.66 /zg), estrone (2.12 /zCi; 12.34 /zg) Present address: University of Michigan School 6 wk later: corticosterone (4.74/zCi; 29.37/zg) of Medicine, Ann Arbor 48103. 8Present address: The Ohio State University, 5 me later: and cort£sol (4.41 /zCi; 30.79 /~g) 3 wk later. The ovariectomized sow was dosed Columbus 43210 4Present address: ]3iologieal Science Research with z4C-cortisol (4.25 /zCi; 29.69 /~g) 4 me after ovarieetomy. Radioactivity in urine was Unit, FBI Laboratory, Washington, DC. 20530. 41
42
J O N E S - W I T T E R S ET AL
indistinguishable from background by 7 days after treatment. Techniques for proof of purity of the hormones, dosing, and rates of excretion in urine have been reported for these injections (4). Urine, collected by urinary catheter, was processed as described by Edgerton ,and Erb (7) and stored at --20 C until analysis. Only urine collected during the fi~rst 0 to 24 h after dosing was used for isolating labeled compounds. General approaches to extraction of free and conjugated steroid metabolites from sow urine, subsequent partial purification and isolation of compounds, and methods of quantification have been published (13, 14, 25, 32). Hydrolyzed urinary estrogen. Urine collected for the Rrst 4 h following injection of estrone and the first 12 h ~ollowing injection of estradiol-17fl was subjected to three successive treatments; enzyme, solvolysis, and acid, to cleave the conjugates from the steroidal aglycons (14). Diethyl ether extraction procedures (4) and separation of extracted compounds into enzyme-neutral, enzyme-phenolic (30), solvolysis-neutral, solvolysis-phenolie, acid-neutral, and acid-phenolic (14) ~ractions was as previously described. Estrogens freed by hydrolysis were separated by thin layer chromatography (TLC) System I which was methanol-methylene chloride (1:20, vol/vol). Location of standards, elution of unknowns &ore zones corresponding to standards, and concentration of metabolites were as previously described (25). Prior to derivative formation the extracts stored in ethanol (--10 C), were dried under vacuum, and the residue was transferred with freshly distilled tetra~ydrofltran (3 X 200/~1) into 1 ml conical tubes. Then the solvent was evaporated under a stream of nitrogen. O-methyloxime (1) and trimethylsilyl ether (13) derivatives were prepared as well as double derivative. For the latter, O-methyloxime was prepared first, the reagent removed with a stream of CO2 with the reaction tube placed in a warm water bath, and then the trimethylsilyl ether (TMS) was prepared. Radioactive compounds were eluted from a gas-liquid chromatography (GLC) column with a 50-50 stream splitter. Steel columns (368 X .32 ern) were packed with 2% liquid phases OV-1 and OV-225 (Supelco, Inc.) coated on Anakrom ABS (80-90 mesh, Analabs, Inc.). Nitrogen and hydrogen flow rates were 25 ml/min. Analytical GI_,C was with a dual column instrument (Varian Aerograph, Model 204-1C). Rentention times were deterJOURNAL OF DAIRY SCIENCE VOL. 58, N o . l
mined relative to 5~-cholestane. A combination gas chromatograph-mass spectrometer (PerkinElmer, Model 270) was used to obtain mass spectral data. Urinary estrogen conjugates. Urine collected from 0 to 8 h after rdC-estrone injection was pooled a~d six 25-ml aliquots were designated samples A through F. Counting aliquots were taken at each step of the procedure to isolate the conjugated estrogens. Free estrogens were extracted from unhydrolyzed or hydrolyzed urine with diethyl ether (1:1, vol/vol). Conjugated estrogens were removed from urine with Amberlite XAD-2 resin (Robin and Haas Co.) by the procedures of Bradlow (2) and Osawa and Slaunwhite (21). Estrogen glucuronides were freed from their conjugating moiety with a fl-glueuronidase (Ketodase, Warner-Chflcott) which does not have sulfatase activity. Glass plates (20 X 20 can) coated with silica gel G (25 mm thick) and three TLC systems were used to separate tmconjugated (System I identified in the preceding section) and conjugated estrogens (Systems II and III). The mixture of conjugates from the XAD-2 resin column were separated in System II (ehloroform:isopropyl ,alcohol:formic acid; 100:60:30, vol/vol/vol) into sulfate, anonoglucuronide, and diconjugate zones. The monoglucuronides were separated in System III into estriol glucuronide and estradiol-estrone glueuronide zones by developing twice in chloroform: methanol: acetic acid (8:1:1, vol/ vol/vol) (27). Mter development, reference s~andards were located (26) and corresponding zones of unknowns were marked on the ehromatograms. Then the silica gel in these zones was scraped onto glassine paper, transferred to test tubes, and 10 ml of solvent was added. The mixture was allowed to stand overnight before filtering through a sintered glass funnel. Chloroform-methanol (1:1 vol/vol) and 50% aqueous methanol were used to elute u nconjugated and conjugated estrogens, respectively. Enzymatic hydrolysis of conjugates and GLC procedures were as reported in the preceding section. Urinary corticold metabolites. Urine voided within 24 h after injection of either x*C-cortisol or 14C-cortieosterone was used to extract the urinary ~*C-labeled metabolites. The procedures initially followed were like those reported for cow urine by Willett et al. (32) except for two differences. During extraction the solvolysis step was unnecessary when fl-glueuronidase (Glusulase, End9 Laboratories) was used for initial hydrolysis. Also, colu~mn chromatography was un-
S T E R O I D METABOLITES
43
TABLE 1. Distribution of extracted radioactivity (~) within sequential cleavage treatments and fractions from urine following injections of a4C-labe]ed estrone and estradiol-17/~. I-lxnrnone injected
Sequential treatment
Fraction Acid"
Phenolic
Neutral
(~) Estrone
Enzymeb Solvolysis
65.3 _ 4.6 ~ .23 _ .05
36.1 - 2.0 background
2.5 ± .3 .24d
Estradiol-17/~
Enzyme Solvolysis
53.3 3.2
38.5 ± 2.4 background
1.4 - .2 .16 ± .02
___ 4.1 ± .66
Extracted into the NaOH/NaCL wash which was at pH 13. Adiustment to pH 3 in subsequent samples resulted in extraction of 79?o of the radioactivity in the acid fraction as result of partitioning at pH 13. b Glusulase, Endo Laboratories. Standard error. d Samples were pooled. necessary as an initial step to separate 14C-labeled compounds due to smaller quantities of pigments in sow urine extracts compared to cow urine extracts. Paper chromatography systems A, C, and D as identified and diagrammed by Willett et ,al. (32) were used to separate urinary radioactive metabolites of aaC-labeled cortisol and cortic~>sterone. Chromatograms of unknowns were observed under UV light to detect a,fl uasaturated ketone structures. Blue tetrazolium and phosphomolybdic acid were used to test for side chains and reactive groups (5). Results and Discussion
Metabolites of estrogen. Totals of 81% and 91% of the radioactivity in urine from injection of ~ C estrone and estradiol-17fi, respectively, were extracted by sequential hydrolysis. Enzyme treatment .alone rendered extractable with diethyl ether, 99.7 ___ 4.3% and 95.4 ± 8.0% of the urinary radioactivity (x of six extractioa~'s) recovered for the estrone and estradiol-17fl iniectious. Due to efficiency of hydrolysis of the crude enzyme mixture, Ghisulase, which contained both sulfatase and ghicuronidase activity, only .31 ± .01% and 4.6 ± .4% of the radioactivity was extracted following solvolysis of urine from the estrone and estradiol-17fl injections. These percentages are s'mailar to those repol~ed by Tarqui et al. (29). The increased amount of r~dioactivity extracted after solvolysis of urine from the estradiol17fl iniection as compared to estrone may represent a minor difference in conjugation pathways between estrone and estradiol-17fi. No additional detectable radioactivity was ether extractable after acid hydrolysh. Thus, it may be unnecessary to use other than Glusulase on
nonpregn.ant sow urine to render estrogens ether extractable. This, however, would need to be checked on additional individuals at different periods of the estrous cycle and during pregnancy to determine the extent of variation between a~_imals. Diethyl ether extracts of urine following enzyme hydrolysis contained more radioactivity in the phenolic fraction than in the acid fraction (Table 1). In eorapaa'ison, enly trace amounts w e r e in the neutral fraction. The data for urine following estradioI-I7fl injection were similar to data for estrone except 3.2% instead of .23% of the radioactivity was in the solvolysis-phenolle fraction (Table 1) which suggests the presence of a sulfate conjugate resistant to Ghisulase. The presence of 36 to 38% of the TABLE 2. Distribution of radioactivity after thinLayer ebxomatography (TLC) of the enzyme-acidic fraction from urine following injection of a4C_,labeled estrone. Collection period
TLC zone system I ~
(h)
(Standard)
0 to 8 pool
Estrone Estradiol-17~ Estriol
4 to 5 pool Estrone Estradiol-17~ Estriol
Radioactivity (No)
(~;)
I
2. Q
II III IV V
36.6 1.7 54.3 4.6
I II III IV V
7.4 15.1 12.4 50.9 14.1
"Zones were numbered starting at the solvent front; 20 X 20 cm plate chromatographed in methanol:methylene chloride ( 1: 9.0, vol/vol). JOURNAL OF D/dRY SCIENCE VOL. 58, NO. I
44
JONES-WlTTERS ET AL
total ~C extracted in the base wash aqueous layer (acidic fraction) was unanticipated and indicated compounds more polar than estriol (19). However, a major portion (79%) of the radioactivity in the acid ~raction was extracted with diethyl ether in subsequent samples after adjusting the NaOH/NaC1 wash from pH 13 to pH 3. About 50% of the radioactivity remaining in the enzyme-acidic fraction a~ter extraction with diethyl ether at pH 3 was in the estriol fraction (Table 2). Approximately 97% of the radioactivity in the enzyme-phenolic fraction (fl~rst six samples extracted) was in the estrone TLC zone (System I) following either estrone or estradiol-17/3 injection. The relatively small .amount of radioactivity in the solvolysis-phenolie fraction from the estrone injection was 100% in the estrone TLC zone. The comparable estradiol-17/3 data showed that, in addition to 91% the radioactivity in the estrone TLC zone, 4.4% and 4.7% of the radioactivity was at the origin and in the estradiol-17fl TLC zone. Samples of the estrone TLC zone from both estrone and estradiol-17fi injections were chromatographed on GLC as trimethylsilyl ether derivatives, and the column effluent was collected for measurement of radioactivity. The estrone peak contained 67% of the radioactivity. The remaining radioactivity, especially in the extract following estrone injection, may have been s',nnilar to an unknown compound previously designated X1 by Jones (12). Even though attempts to identify this compound were unsuccessful, the compound was not a contaminant of r'C-labeled compounds injected. Another unidentified peak, accounting for 10% of the radioactivity chromatographed, was eluted from urinary extracts following injection of estrone but not estradiol. As will be shown later, 21% of the radioactivity was in the same peak following fl-glucuronidase treatment and TLC (System III) of the phenolic glucuronide fraction of extract from the estrone injection. The compound may be the 2-methoxyestrone identified by Terqui (28). Mass spectra were obtained of standard estrone TMS, and of the TMS derivatives of endogenous estrone and X1 isolated from extracts of urine after estrone injection as each of the compounds ~vas eluted from an OV-1/OV-225 column. The spectrum of endogenous estrone correlated with standard estrone and with published spectra (20). The spectrum of X1 TMS could not be identi~ed as a steroid. In associated work on pregnant sows, Edgerton and Erb (7) reported one unknown comJOURNAL OF DAIRY SCIENCE VOL. ~8, NO. 1
pound identical in GLC properties to estradiol17fl and another unknown with GLC properties similar to estradiol-17~. Mass spectral analysis of the estradiol TLC zone of day 103 pregnancy urine from the sows mentioned above verified the GLC identifi~Cation of estradiol-17fl by Edgerton and Erb (~/). Comparison of the acetylated total phenolic extract (7) with acetylated TLC zone extracts indicated that X1 probably was the compound with GLC properties similar to the acetate derivative of standard estradiol-17a. Urinary estrogen con/ugates. Each of the six 25-ml replicate samples contained 19,100 __ 379 dpm. The distribution of radioactivity after extraction of the urine with diethyl ether showed that 95.0 __ .9% of the labeled estrone either had been conjugated or metabolized and conjugated by the sow and remained in the aqueous layer. Only 1.8 ± .2% was ether extractable prior to hydrolysis as compared to 14.5% from urine of ,a pregnant sow (16). The lower value may reflect differences in physiological state and dosage. Estrogen conjugates are compounds of the type absorbed by XAD-2 resin which can be eluted by the polar alcohols, ethanol and methanol. Almost all of the r~dioactivity (91.8 ± 1.1%) was in these conjugates. The first fraction, eluted with ethanol, contained 71.1 ± 2.2% of absorbed radioactivity; and the second fraction, eluted with methanol, contained 22.9 ± 1.2%. The remainder was essentially accounted ~or in the water wash. When these two alcoholic fractions were processed separately, the percentage distribution of radioactivity by TLC zone following chromatography of conjugated and nonconjugated compounds was almost identical. Therefore, the ethanol and methanol fractions were combined .as the alcoholic fraction for further analyses. After the alcoholic extracts from Sample C were hydrolyzed with Ketodase to free the estrogens present as glucuronides, 96 ± 3% of the 1~C was extracted with diethyl ether. The compounds extracted into ether were chromatographed in System I. The estrone zone contained 84.4% of the radioactivity, and ,approximately 9% of the total dpm were in the estriol and adjacent less polar TLC zones. Alcoholic fraction,s from samples A, B, and D to F contained 93.3 +__ 2.1% of the ~*C in the monoglucuronide zone and only 5.6 ± 2.1% in the diconjugate zone (Table 3). Only .8 ± .2% of the 14C was in the sulfate zone and only .3 ± .1% at the origin. These results are
S T E R O I D METABOLITES
45
TABLE 3. Distribution of radioactivity (%) folio,wing thin-layer chromatography (System II) ~ of the conjugates eluted from amberlite XAD-2 by ethanol and methanol.
Sample
Zone I (origin)
Zone II ( diconiugates )
A B D E F Mean
.2 .5 .2 .6 .2 .3 - .1b
1.2 1.5 4.2 10.2 10.8 5.6 ± 2.1
Zone III (monoglucuronides) 98.2 97.2 94.1 88.2 88.6 93.3 + 2.1
Zone IV (monosulfates, solvent front) .4 .8 1.5 1.0 .4 .8 _-2 .2
Chloroform:isopropyl alcohol:formic acid ( 100:60:30 ), ( vol/vol/vol ). b Standard error. in marked contrast to those of Kubomichi (16) who reported 21.2% of the total estrogens in the sulfate fraction 'and 64.3% in the glucuronide fraction. However, the sulfate fraction was obtained by acid hydrolysis at 32 C with simultaneous extraction into ether, and the glueuronide fraction by vigorous ,acid hydrolysis after removal of the sulfates. These methods have caused structural alterations, and Kubomichi (16) recommended that future research use enzymatic rather than acidic hydrolysis. The monogtueuronide zone from Sample B was rechromatographed in System Ili. The estriol-3-glucuronide zone contained 8.2%, and the estradiol-17-glueuronide-estrone-3-glucuronide zone contained 87.6% of the radioactivity. When the TLC monoglucuronide zones from Samples D and E were hydrolyzed by Ketodase and extracted with diethy1 ether, more than 98% of the radioactivity was in the organic phase. When the concentrated diethyl ether extracts were chromatographed in System I, 94.2 4- 1.0% of the label was in the estrone zone, 2.1 ___ 0.6% was in the estriol zone, and 1.9 ± 0.1% was in the estr~diol zone. The TMS derivatives of compounds: in the estrone TLC zone from Samples D and E above were chromatographed by GLC as described earlier. Four peaks were resolved from the biological material. The relative retention
times (RRT) of these peaks were based on 5~eholestane, which had a retention time of 15.2 rain. Of the radioactivity ehromatographed, peak 1, .an unknown (RRT .52), contained 8.1%; peak 3, estrone (RRT .80), contained 65.4%; and peak 4, an unknown (RRT .89), contained 21.3% of the radioactivity chromatographed. These results are in fair agreement with results cited in the preceding section for estrogens extracted after hydrolysis and separated in the same TLC system. Peak 4 may be 2-methoxyestrone, ~dentit%d in the urine of the pregnant sow following injection of labeled estrone and estradiol (28). Urinary corticoid metabolites. The percentage ef 14C extracted with ethyl ,acetate before enzyme hydrolysis accounted for only 4.6 to 9.4% of the total radioactivity (Table 4). More than 60% was extracted following enzyme hydrolysis (Glusulase) of urine from cortisol injections as compared to about 50% for cortieosterone. From 16 to 28% of the radioactivity was not extracted, and an additional 9 to 17% was unaccountable loss. As shown from preliminary extractions, only an additional 1 to 4% could have been extracted by including solvolysis as used by Willett et al. (32). Radioactivity extracted before enzyme hydrolysis and after solvolysis was too low ~or characterization of compounds in the respective extracts.
TABLE 4. Average percentage of 14C extracted before and after enzyme hydrolysis of sow urine voided after injection of ~4C eortisol and cortieoateroneL Sow Ovariectomized Intact Intact
14 C compound Cortisol Cortisol Cortieosterone
Before
Hydrolysis After
Total extracted
4.6 ± .4b 6.9 ± .6 9.4 ± 2.0
69.9 ± 1.9 60.9 ± 2.6 49.4 --- 3.4
74.5 --- 2.8 67.8 ± 3.1 58.8 ± 3.1
* Each sample prior to extraction contained approximately 1.0 X 105 dpm. ~' Standard error; n = 12. JOURNAL OF DAIRy SCIENCE VOL. 58, NO. 1
46
JONES-WITTERS ET AL
TABLE 5. Distribution of extracted a~C (average ~) among urinary corticoid rnetabolites isolated by paper chromatography systems A, C, and D as described by Willett et al. (32). Compounds ~
Ovariectomized Cortisol
Intact Cortisol
Cortieosterono
(~) 6 hydroxycortisol Cortol Cortolone Tetrahydroeortisol Tetrahydrocortisone Cortisol Corticosterone Tetrahydroeortieosterone 11-ketoetiocholanolone and 11/S-hydroxyetiocholanolone
2.2 11.6 21.4 23.8 30.8 5.9 .3 3.4
_.+ _ _-. __. ± ± --±
13.0 ±
1.3~ 4.9 3.0 4.1 10.7 2.9 .2 1.0 2.4
7.0 13.5 11.7 43.2 8.8 6.9 .7 .5
__+ 2.9 ___ 2.8 _ 2.0 ± 9.2 __. 2.0 +__ 3.3 ± .7 _ .3
8.6 ± 3.9
1.3 11.7 5.6 2.7 7.7 0.5
--+ .9 ± 4.6 _ 1.9 + 1.~ ± 7.1 - .3
20.5 +-. 3.7
31.6 ± 8.2 18.5 ± 5.0
Standard error; n = 9. A minimum of nine compounds was isolated from extracts of enzyme hydrolyzed urine (Table 5). The maior urinary metabolites of cortisol corresponded to chromatographic properties of tetrahydrocortisol (THF) and tetrahydroeortisone (THE) standards. Although THF was the major metabolite in the intact sow, totals of T H F and THE were similar, averaging 54.6% and 52.0% for the ovariectomized and intact sows and similar to data for human beLugs (11, 23). In contrast, both T H F and T H E were low in urine of the intact sow following injection of corticosterone. For the corticosterone injection, major urinary rnetabolites corresponded to the isolated eorticosterene and tetrahydrocorticosterone (THB) standards. These two compounds ,accounted ~or 52% of the radioactivity isolated from extracts of urine from the eortieosterone injection. These results agree with similar data for human beings (22) and cows (32). The urine of both sows contained considerable radioactivity in areas of chromatogrr~ms corresponding to cortol, cortolone, and ll-ketoetiocholanolone-llfl-hydroxyetiocholanolone standards (Table 5). General Discussion
Estrogens. The estrone ~raction of urine from the sow contains the largest amount of estrogen metabolite, and the amount of estradiol17/3 is low .as previously reported (17, 29, 31). Additionally, the loss of a large percentage of radioactivity not in estrene or estradiol-17fl fractions ,agrees with Terqui et a]. (29). The evidence, although inconclusive, favors a small a~ount of an estriol-like compound in sow urine which also tras been reported by others
(3, 19, 28). In the nonpregnant sow estrone is eonjugatJouaNaL OF DAIRYSCI~NCSVOL. 38. NO. 1
ed primarily as the monoglucuronide (Table 3). Terqui et al. (29), using the Brown method of separation, found that when a sow was injected with either labeled estrone or estradiol, the bulk of the label was recovered in the urine in the estrone fraction. Lunaas (17), also using Brown's method of separation, found that estrone was the major estrogen recoverable ~rom sow urine, both during early pregnancy and during the estrons cycle. However, he discarded the portion of urine extract which would have contained estriol. Kubomichi (16) and Bredeck and Mayer (3) reported that estriol was the major urinary estrogen in pregrrant sows in contrast to the 98% estrone reported by Edgerton and Erb (7) for day 110 of pregnancy. Edgerton and Erb (7) used enzyme hydrolysis during extraction and quantification by gas liquid chromatography, as compared to acid hydrolysis and quantification by colorimetry (3, 16). Kubomichi (16) attempted to identify the types of conjugates by differential acid hydrolysis. He found that 83% of the estrogens were conjugated as ghicuronides and 21% were conjugated as sulfates. This does not agree with our ~ndings since over 95% of the 1'4C was in the glucuronide fraction. However, differences in conjugate isolation technique and reproductive status of the sows probably account for the different results. Incomplete hydrolysis and desta'uction of aglycones may be avoided by extraction of the estrogen conjugates with XAD-2 resin rather than by differential acid hydrolysis (21). Dtffour and Raeside (6) have suggested that estrogen formation occurs largely in the placenta of the pregnant sow, so that the percentage estrogen conjugated ,as sulfate may well differ from that in urine of the nonpreg-
STEROID METABOLITES
nant so~v. If it is later verified in a larger population of sows that urinary estrone of sows is conjugated mostly as the monoghmuronide, then quantitative methods can be greatly simplified by using only enzyme hydrolysis and substitution of radioimmunoassay for GLC quanti~cation. Corticoids. T h e sow excretes in urine 80 to 90% of the radioactivity injected as zdC-labeled cortisol and corticosterone (4) as compared to 20 to 29% for the cow (33). The majority of corticoid metabolites in sow urine were similar to the parent compounds relative to containing 21 carbons (9) and were reduced in the A ring to tetrahydro derivatives (10) of THF, THE, and THB. The presence of a larger quantity of T H E (30.8%) than T H F (23.8%) in urine of the ovariectomized sow (Table, 5) was about the same as reported for the human being (9, 9,3). The predominance of T H F (43.2%) rather than T H E (8.8%) in the urine of the intact sow after eortisol injection was similar to changes associated with disease induced stress in the human being (11). Therefore, the intact sow may have had a stress reaction when cortisol was administered. Willett and Erb (34) observed a marked inerease in plasma corticoids when 14C-labeled cortisol was adininistered to a heifer. Also, plasma cortisol was increased in heifers as much as five times from psychological stimuli (34). In general, it appears that the primary urinary route of excretion (4) and the major urinary metabolites of cortisol and corticosterone (Table 5) in the sow are typical of results for the human being (9, 11, 22, 23). Acknowledgments The authors thank L. A. Edgerton, Smith Kline Laboratories, £or administration of estrogens and collection of urine while at Purdue University; Philip Gill, E. I. du Pont de Nemours and Co., for gas chromatography-mass spectrometry, W. W. l~audler, Department of Chemistry, Ohio University, for interpretation of the mass spectra; and W. C. Afford and W. Klyne for sodium estradiol-3-glueuronide, sodium estradiol-17fl-glucuronide, sodium estriol3-glucuronide, sodium estriol-18a-glucuronide, sodium estriol-17fl-glucuronide, 16a-hydroxyestrone- 16~-glucuronide, 16fl-hydroxyestrone16fl-glucuronide, and sodium estrone-3-glucuronide from the MRC Steroid Reference Collection. References (1) Allen, J. G., C. H. Thomas, C. J. W. Brooks, ,and B. A. Knights. 1969. The determina-
47
tion of stereochemistry at C-5 of 3, 6-dioxygermted steroids. Steroids 13:133. (2) Bradlow, It. L. 1968. Extraction of steroid conjugates with neutral resins. Steroids 11: 265. (3) Bredeck, H. E., and D. T. Mayer. 1958. Estrogenic steroids in swine pregnancy urine. Page 157 in E. X. Gassner, ed., Reproduction and infertility, III Symposinrn. Pergamon Press, New York, NY. (4) Brown, B. L., L. A. Edgerton, L. B. Willett, R. D. Randel, T. G. Dunn, and R. E. Erb. 1970. Excretion of 14C in urine of the domestic sow after inieetion of radioactive estradiol-17fl, estrone, cortieosterone and cortisol. J. Anita. Sci. 31:1186. (5) Domingnez, O. V. 1967. Chromatography of steroids on paper. Page 135 in H. Carstensen, ed., Steroid hormone analysis, Vol. 1. Marcel I)ekker, Inc., New York, NY. (6) Dufour, J., and J. I. Raeside. 1969. Hydroxysteroid dehydrogenase activity in the placenta of the domestic pig. Endocrinology 84:426. (7) Edgerton, L. A., and R. E. Erb. 1971. Metabelites of progesterone and estrogen in domestie sow urine. I. Effect of pregnancy. J. Anita. Sci. 32:515. (8) Frantz, A. G., F. H. Katz, and J. W. Tater. 1961. 6/3-hydroxycortisol and ocher polar corticosteroids: Measurement and significance in human urine. J. Clin. Endoerinol. Metab. 21:1290. (9) Fukushima, D. K., H. L. Bradlow, L. Hellman, B. Zumoff, and T. F. Gallagher. 1960. Metabolic transformation of hydrocortisone4JdC in nonnal men. J. Biol. Chem. 235: 2246. (10) Gold, N. I. 1961. Partial chara~evizatlon of the metabolites of cortisol-4-1~C in the dog. I. The intact dog. J. Biol. Chem. 236:1924. (11) Gray, C. H., and D. A. Shaw. 1965. The metabolism of (4-~'C) cortisol in patients with collagen diseases. J. Endocrinol. 33:33. (12) Jones~ P. H. 1967. Isolation, identification ,and measurement of metabolities of progestins and estrogens in the urine of donaestie sows during the estrous cycle. Ph.D. Thesis. Purdue University, West Lafayette, IN. (13) Jones, P. H., and R. E. Erb. 1967. Modified procedure for estimating estrogens in urine. J. Dairy Sci. 58:772. (14) Jones, P. H., and B. E. Erb, 1968. Comparison of metabolites of progesterone-14J~C in urine from a nonprognant domestic sow. J. Anita. Sci. 27:1049. (15) Jones-Witters, P., and R. Phillips. 1971. Metabolites of ~C-labeled estrogens in the sow. J. Anita. Sci. 33:1159. (Abstr.) (16) Kubornichi, M. 1966. Studies on the m-inary estrogens of pregnant sows. Nat. Inst. Anita. Health Quart. 6:33. JOURNAL OF DAIRY SCIENCI~ VOL. ~8, NO. 1
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JONES-WITTERS E T AL
(17) Lunaas, T. 1962. Urinary estrogen levels in the sow during estrous cycle and early pregnancy. J. Reprod. Fert. 4:13. (18) Mellin, T. N., and R. E. Erb. 1966. Estrogen metabolism and excretion during the bovine estrous cycle. Steroids 7:589. (19) Mellin, T. N., R. E. Erb, and V. L. Ester,green. 1965. Quantitative estimation and identification of estrogens in bovine urine. J. Dairy Sci. 48:895. (20) Okerholm, R. A., S. I. Clark, and H. H. Wotiz. 1971. Effect of derivative formation on the mass spectra of the estrogens. Anal. Biochem. 44:1. (21) Osawa, Y., and W. R. Slaunwhite, Jr. 1970. Studies on phenolic steroids in human subjects. XIII. A rapid assay of urinary conjugates in pregnancy. Steroids 15:73. (22) Peterson, B. E., and C. E. Pierce. 1960. The metabolism of eortieosterone in man. J. Clin. Invest. 39:741. (23) Romanoff, L. P., C. W. Morris, P. Welch, R. M. Rodriquez, and G. Pineus. 1961. The metabolism of cortisol-4-1'C in young and elderly men. L Secretion rate of eortiso.1 and daily excretion of tetrahydroeortisol, allotetrahydroeortisol, tetrahydroeortisone and cortolone (20a and 20~). J. Clin. Endoerinol. Metab. 21:1413. (24) Schomberg, D. W., W. R. Featherston, and R. E. Erb. 1965. Excretion of metabolites of progesterone-4-a~C in the cycling sow. J. Anim. Sei. 27:1195. (25) Sehomberg, D. W., P. H. Jones, W. R. Featherston, and R. E. Erb. 1966. Identification of metabolites of progesterone-4-~4C in domestic sow urine. Steroids 8:277.
JOURNAL OF DAIRY SCIENCE VOL. 58, N O . I
(26) Stahl, E., ed. 1965. Thin-layer chromatography. Academic Press, Inc., New York, NY. (27) Takriti, N., and W. Thorn. 1965. Separation and detection of gluenronides. Angew. Chem. Intern. Ed. Eng. 4:1095. (28) Terqui, M. 1971. Estrogen metabo//sm in the pregnant sow. I. Urinary estrogens. Ann. Biol. Anita. Biochem. Biophys. 11:569. (29) Terqui, M., P. Rombauts, and J. Fevre. 1968. Comparative fecal and urinary excretion of estrogens in the ewe and sow. Ann. Biol. Anita. Biochim. Biophys. 8:339. (30) TiUson, S. A., R. E. Erb, and G. D. Niswender. 1970. Comparison of luteinizing hormone and progesterone in blood and metabolites of progesterone in urine of domestie sows during the estrous cycle and early pregnancy. J. Anita. Sei. 30:795. (31) Velle, W. 1959. Isolation of oestrone from the tu-ine of the pregnant sow. Aeta. Vet. Scan& 1:19. (32) WiUett, L. B., B. L. Brown, and R. E. Erb. 1972. Isolation of metabolites of 4-a4C-cortieosteroids from urine of an ovarieetomized heifer. J. Dairy Sci. 55:65. (33) Willett, L. B., T. G. Dunn, B. L. Brown, and R. E. Erb. 1971. Excretion of 4-~C eortieosteroids in urine by an ovarieetomized bovine. J- Anita. Sci. 32:119. (34) Willett, L. B., and R. E. Erb. 1972. Short term changes in plasma cortieoids in dairy cattle. J. Anita. Sci. 34:103. (35) Williams, W. F. 1962. Excretion of progesterone and its metabolites in milk, urine and feces. J. Dairy Sei. 45:1541.
